Journal: Nature materials

Article Title: A Year-Long Extended Release Nanoformulated Cabotegravir Prodrug

doi: 10.1038/s41563-020-0674-z

Figure Lengend Snippet: ( a ) CAB was chemically modified with 14, 18, and 22 carbon fatty acid chains to develop MCAB, M2CAB, and M3CAB, respectively. ( b ) FTIR spectra show the presence of absorption bands around 2,919 cm −1 and 1,765 cm −1 corresponding to C-H stretch in fatty acid methylene groups and carbonyl stretch, respectively. Data was independently reproduced three times. ( c ) Aqueous solubility of CAB and prodrugs. Data are expressed as the mean ± SEM for N = 3 (CAB), 3 (NMCAB), 3 (NM2CAB), and 3 (NM3CAB) biologically independent samples. t test (two-tailed) with Welch’s correction was used to compare the solubility between CAB and individual prodrug. (*P < 0.05). ( d ) Antiviral activity was determined in MDM over a range of concentrations (0.01 – 1,000 nM) by measuring HIV-1 RT activity after challenge with HIV-1 ADA at an MOI of 0.1. Data are expressed as the mean ± SEM for N = 3 biological replicates. ( e ) Plasma cleavage kinetics. Bioconversion of prodrugs into active CAB in plasma of various species (mice, rat, rabbit, monkey, dog, and human) was assessed. Experiments were repeated independently two times with equivalent results.

Article Snippet: Four male RM (4.4–6.7 kg; PrimeGen, Santa Ana, CA) were anesthetized with ketamine (10 mg/kg); each was administered 45 mg/kg CAB-equivalents of NM2CAB and a lab-generated RPV prodrug nanoformulation by IM injection (quadriceps femoris muscle, 0.5 mL/kg).

Techniques: Modification, Solubility, Two Tailed Test, Activity Assay

Journal: Nature materials

Article Title: A Year-Long Extended Release Nanoformulated Cabotegravir Prodrug

doi: 10.1038/s41563-020-0674-z

Figure Lengend Snippet: ( a ) Morphological assessment of NCAB, NMCAB, NM2CAB, and NM3CAB by SEM. Data was independently reproduced three times. ( b ) The stability of CAB and prodrug nanoformulations was evaluated over 98 days following manufacture at room temperature, 4 ºC, and 37 ºC as determined by size (nm), polydispersity index (PDI), and zeta potential (mV). Data are expressed as the mean ± SEM for N = 3 biological replicates.

Article Snippet: Four male RM (4.4–6.7 kg; PrimeGen, Santa Ana, CA) were anesthetized with ketamine (10 mg/kg); each was administered 45 mg/kg CAB-equivalents of NM2CAB and a lab-generated RPV prodrug nanoformulation by IM injection (quadriceps femoris muscle, 0.5 mL/kg).

Techniques:

Journal: Nature materials

Article Title: A Year-Long Extended Release Nanoformulated Cabotegravir Prodrug

doi: 10.1038/s41563-020-0674-z

Figure Lengend Snippet: ( a ) Cellular uptake and ( b ) retention of drug and each prodrug in MDM was assessed. ( c ) In parallel, CAB released into the culture medium was measured. For uptake study, a one-way ANOVA followed by Tukey’s post-hoc test was used to compare CAB levels among four treatment groups (red colored ## P < 0.01, ***P < 0.001, $ $ $ $ P < 0.0001 compared to NCAB), and prodrug levels among three treatment groups (blue colored @ P < 0.05 compared NMCAB). For retention study, a one-way ANOVA followed by Tukey post-hoc test was used to compare CAB levels among four treatment groups (red colored $ $ $ $ P < 0.0001, #### P < 0.0001 compared to NCAB), and prodrug levels among three treatment groups (blue colored @@ P < 0.01, ^^^P < 0.001 compared to NMCAB, and green colored ^^P < 0.01 compared to NM2CAB). For release study, a one-way ANOVA followed by Tukey’s post-hoc test was used to compare CAB levels among four treatment groups (red colored *P < 0.05). For all assays, data are expressed as mean ± SEM, N = 3 biological replicates. ( d ) TEM images of MDM treated with each nanoformulation and of untreated control. Red arrowheads indicate nanocrystals present in the cytoplasm. Scale bars = 2 μm. Data was independently reproduced three times. ( e ) Antiretroviral activity. Infection was determined by measuring HIV-1 RT activity in culture medium; and ( f ) HIV-1 p24 antigen immunocytochemistry. ( g and h ) The antiretroviral concentration-response of NM2CAB. ( e and g ) HIV-1 RT activity was expressed as mean ± SEM, N = 4 biological replicates. ( f and h ) Each experiment was repeated independently three times with equivalent results.

Article Snippet: Four male RM (4.4–6.7 kg; PrimeGen, Santa Ana, CA) were anesthetized with ketamine (10 mg/kg); each was administered 45 mg/kg CAB-equivalents of NM2CAB and a lab-generated RPV prodrug nanoformulation by IM injection (quadriceps femoris muscle, 0.5 mL/kg).

Techniques: Activity Assay, Infection, Immunocytochemistry, Concentration Assay

Journal: Nature materials

Article Title: A Year-Long Extended Release Nanoformulated Cabotegravir Prodrug

doi: 10.1038/s41563-020-0674-z

Figure Lengend Snippet: Tissue and blood concentrations of prodrugs (MCAB or M2CAB) were determined at 14, 28, 42 and 364 days after a single IM injection of NMCAB or NM2CAB. Prodrug levels were measured in the ( a ) spleen, ( b ) liver, ( c ) gut, ( d ) lung, ( e ) brain, ( f ) rectal tissue, ( g ) kidneys, ( h ) lymph nodes-anatomical associated tissues, and ( i ) blood. Prodrug levels in lymph nodes were determined in anatomical regions associated with lymph nodes only at days 28 and 364, due to their immature state in immunodeficient NSG mice. ( a-h ) Data are expressed as mean ± SEM. For days 14, 28, and 42 groups, animal numbers in each group were N = 5, and for the day 364 group animal numbers were N = 3 (NMCAB), and N = 4 (NM2CAB). t test (two-tailed) with Welch’s correction was used to compare prodrug levels in tissues at the respective time points (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). ( i ) Data are expressed as mean ± SEM. Study was initiated with N = 5 animals in each group (NCAB, NMCAB or NM2CAB). Due to loss of animals by natural causes during study period, by the day 364 post-treatment, N = 5 (NCAB), N = 3 (NMCAB) and N = 4 (NM2CAB) animals.

Article Snippet: Four male RM (4.4–6.7 kg; PrimeGen, Santa Ana, CA) were anesthetized with ketamine (10 mg/kg); each was administered 45 mg/kg CAB-equivalents of NM2CAB and a lab-generated RPV prodrug nanoformulation by IM injection (quadriceps femoris muscle, 0.5 mL/kg).

Techniques: Injection, Two Tailed Test

Journal: Nature materials

Article Title: A Year-Long Extended Release Nanoformulated Cabotegravir Prodrug

doi: 10.1038/s41563-020-0674-z

Figure Lengend Snippet: Four rhesus macaques were administered a 45 mg/kg CAB-equivalent dose of NM2CAB by a single IM injection. ( a ) Plasma samples were collected, and CAB and M2CAB levels were determined up to day 365. ( b and c ) Rectal, lymph node, and adipose tissue biopsies were collected at day 204 following drug administration and assayed for ( b ) CAB and ( c ) M2CAB concentrations. ( d and e ) Systemic adverse reactions were evaluated by measuring ( d ) hematologic and ( e ) metabolic profiles. Plasma drug and prodrug concentrations, and hematologic and metabolic profile parameters are shown for individual animals. Tissue drug concentrations are expressed as mean ± SEM; N = 4 animals.

Article Snippet: Four male RM (4.4–6.7 kg; PrimeGen, Santa Ana, CA) were anesthetized with ketamine (10 mg/kg); each was administered 45 mg/kg CAB-equivalents of NM2CAB and a lab-generated RPV prodrug nanoformulation by IM injection (quadriceps femoris muscle, 0.5 mL/kg).

Techniques: Injection

Journal: International Journal of Molecular Sciences

Article Title: Synthesis of Fe 3 C@C from Pyrolysis of Fe 3 O 4 -Lignin Clusters and Its Application for Quick and Sensitive Detection of PrP Sc through a Sandwich SPR Detection Assay

doi: 10.3390/ijms20030741

Figure Lengend Snippet: ( A ) SPR response of PrP Sc with concentrations of 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL, respectively. Insert: Calibration curve of Fe 3 C@C-aptamer involved SPR detection assay. ( B ) Specific analysis of Fe 3 C@C-aptamer involved SPR amplification detection. 1: PrP Sc (10 ng/mL) in PBS buffer; 2: PrP Sc (10 ng/mL) in NBCS; 3–6: MPA, thioPEG, Cys-protein G and PrPC (10 ng/mL each) in PBS buffer, respectively; 7–10: Mixture of PrP Sc (10 ng/mL) and each of the four different reagents (10 ng/mL) in PBS buffer.

Article Snippet: The Cys-protein G (Catalog #: 1002-04) was obtained from Shanghai PrimeGene Bio-Tech Co. in China.

Techniques: Detection Assay, Amplification

Journal: Frontiers in Immunology

Article Title: Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell

doi: 10.3389/fimmu.2021.697071

Figure Lengend Snippet: rhHMGB1-induced endothelial barrier hyperpermeability and RhoA/ROCK1 expression in ECs. (A) Cell viability of HPMEC was evaluated by CCK-8 measurement after stimulated with different concentration of rhHMGB1 for 24 h. (B) HPMECs were stimulated with the indicated concentrations of rhHMGB1 for 24 h. (C) HPMECs were stimulated with 600 ng/ml rhHMGB1 for the indicated times. (D, E) Immunofluorescence location of F-actin, VE-cadherin and ZO-1 in HPMECs was detected after 600 ng/ml rhHMGB1 stimulation for the indicated times. The fluorescence intensity of F-actin, VE-cadherin and ZO-1 was quantitatively analyzed using the Image J software. (F) The concentration of 600 ng/ml rhHMGB1 could selectively downregulate the expression level of VE-cadherin and ZO-1 at 24 h. (G) Time course of rhHMGB1-mediated increase in RhoA activity. Western blots showed the content of GTP-bound RhoA and total RhoA in cell lysate. (H) rhHMGB1 (600 ng/ml) treatment significantly upregulated ROCK1 expression in HPMECs at 60 min. (I) Treatment with 600 ng/ml rhHMGB1 could transiently promote the expression of pMLC. Values were shown as mean ± SD of 3 independent trials. * p < 0.05 vs . control.

Article Snippet: Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China).

Techniques: Expressing, CCK-8 Assay, Concentration Assay, Immunofluorescence, Fluorescence, Software, Activity Assay, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell

doi: 10.3389/fimmu.2021.697071

Figure Lengend Snippet: Y-27632 pretreatment attenuated rhHMGB1-induced ROCK1/pMLC expression. (A) CCK-8 assay was performed with HPMECs for 24 h with different dosages of Y-27632 as indicated. (B) Effects of Y-27632 on changes in FITC-dextran flux in HPMECs. HPMECs were pretreated with Y-27632 and then treated with 600 ng/ml rhHMGB1 for 60 min. (C) Role of Y-27632 pretreatment in increased barrier permeability induced by rhHMGB1 at 24 h. (D) Effects of Y-27632 on rhHMGB1-mediated morphological change in endothelial F-actin, VE-cadherin and ZO-1. HPMECs were pretreated with Y-27632 for 1 h before rhHMGB1 (600 ng/ml) stimulation for 60 minutes to examine morphology of endothelial F-actin, VE-cadherin and ZO-1 by immunofluorescence. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Y-27632 pretreatment downregulated the ROCK1 expression induced by rhHMGB1 at 60 min. (F) Effects of Y-27632 treatment on rhHMGB1-induced changes in the protein expression levels of VE-cadherin and ZO-1 at 24 h. Y-27632 were added for the last 4 h of the 24 h rhHMGB1 treatment. (G) Pretreatment with Y-27632 attenuated rhHMGB1-induced MLC phosphorylation at 60 min. ECs were pretreated with Y-27632 for 1 h and then stimulated with rhHMGB1 (600 ng/ml) for 1 h. Values were indicated as mean ± SD of 3 separate trials. * p < 0.05 vs . control. # p < 0.05 vs . rhHMGB1 60-min group.

Article Snippet: Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China).

Techniques: Expressing, CCK-8 Assay, Permeability, Immunofluorescence, Fluorescence, Phospho-proteomics, Control

Journal: Frontiers in Immunology

Article Title: Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell

doi: 10.3389/fimmu.2021.697071

Figure Lengend Snippet: FPS-ZM1 treatment alleviated rhHMGB1-mediated RhoA/ROCK1 activation and barrier dysfunction. (A) Cell viability was determined by CCK-8 assay after treated with different dosage of FPS-ZM1 for 24 h. (B) FPS-ZM1 improved lung endothelial permeability at 60 min and 24 h after rhHMGB1 stimulation. (C) FPS-ZM1 significantly downregulated RhoA and ROCK1 expression in HPMECs at 60 min after rhHMGB1 stimulation. (D) Effects of FPS-ZM1 on rhHMGB1-mediated morphological changes in endothelial F-actin, VE-cadherin and ZO-1. ECs were treated with FPS-ZM1 for 1 h prior to stimulation with rhHMGB1 to evaluate morphology of endothelial cytoskeleton F-actin or for the last 4 h of the 24 h rhHMGB1 stimulation to assess morphology of endothelial VE-cadherin and ZO-1 by immunofluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (E) FPS-ZM1 significantly increased VE-cadherin and ZO-1 expression levels in HPMECs at 24 h after rhHMGB1 stimulation. ECs were treated with FPS-ZM1 for the last 4 h of the 24 h rhHMGB1 stimulation. (F) Effects of FPS-ZM1 on rhHMGB1-mediated pMLC expression in cells. ECs were pretreated with FPS-ZM1 for 1 h and then treated with rhHMGB1 for 60 minutes. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . control. # p < 0.05 vs . rhHMGB1 60-min group or rhHMGB1 24-h group.

Article Snippet: Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China).

Techniques: Activation Assay, CCK-8 Assay, Permeability, Expressing, Immunofluorescence, Microscopy, Software, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell

doi: 10.3389/fimmu.2021.697071

Figure Lengend Snippet: rhHMGB1-mediated early barrier dysfunction is largely dependent on ROCK1 signaling. (A, B) ECs were transfected with ROCK1/2 siRNA. Western blot was used to assess the protein expression of ROCK1/2 in HPMECs. (C) Cell viability was assessed by the CCK-8 assay after transfection with different concentration of ROCK1/2 siRNA for 24 h or transfection with 10 nM ROCK1/2 siRNA for the indicated times. There was no evidence of cytotoxicity found in ROCK1/2 siRNA transfected cells. (D) Examination of FITC-dextran flux of HPMECs. ROCK1 knockdown ameliorated rhHMGB1-induced early permeability increases (at 60 min). (E) Effects of ROCK1 siRNA on the expression of VE-cadherin and ZO-1 induced by rhHMGB1 at 24 h. (F) ECs were transfected with ROCK1 siRNA and then stimulated with rhHMGB1 for 60 min. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was detected by fluorescence microscopy. Image J software was used to analyze the fluorescence intensity of F-actin, VE-cadherin and ZO-1. (G) ROCK1 knockdown attenuated the ROCK1 expression induced by rhHMGB1 at 60 min. (H) ROCK1 knockdown downregulated the rhHMGB1-induced pMLC expression in cells at 60 min. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . corresponding control group. # p < 0.05 vs . rhHMGB1 60-min group. NC, negative control.

Article Snippet: Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China).

Techniques: Transfection, Western Blot, Expressing, CCK-8 Assay, Concentration Assay, Knockdown, Permeability, Immunofluorescence, Staining, Fluorescence, Microscopy, Software, Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell

doi: 10.3389/fimmu.2021.697071

Figure Lengend Snippet: rhHMGB1-induced RhoA/ROCK1 pathway activation via RAGE in HPMECs. (A) ECs were transfected with RAGE siRNA. Western blots were used to determine the expression of RAGE in endothelial cells. (B) Cytotoxicity of RAGE siRNA was assessed by CCK-8 assay after transfected with different concentration of RAGE siRNA for 24 h or transfected with 100 nM RAGE siRNA for the different times. No evidence of cytotoxicity was found in RAGE siRNA transfected cells. (C) Treatment with RAGE siRNA ameliorated endothelial barrier dysfunction induced by rhHMGB1 at 60 min and 24 h. (D) ECs were transfected with RAGE siRNA and then stimulated with rhHMGB1 for 60 min and 24 h. Immunofluorescence staining of F-actin, VE-cadherin and ZO-1 was determined by fluorescence microscopy. Fluorescence intensity of F-actin, VE-cadherin and ZO-1 was measured in ECs. (E) Knockdown of RAGE by siRNA reduced the rhHMGB1-induced MLC phosphorylation at 60 min as detected by western blot. (F) Effects of inhibition of RAGE with siRNA on increased expression of RhoA and ROCK1 induced by rhHMGB1 at 60 min in HPMECs. (G) Role of RAGE siRNA in the VE-cadherin and ZO-1 protein expression levels in ECs at 24 h after rhHMGB1 treatment. Mean ± SD of 3 independent trials was shown. * p < 0.05 vs . corresponding control group. # p < 0.05 vs . rhHMGB1 60-min group or rhHMGB1 24-h group. NC, negative control.

Article Snippet: Recombinant human HMGB1 (rhHMGB1) was obtained from Shanghai Primegene Bio-Tech Co., Ltd (Shanghai, China).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, CCK-8 Assay, Concentration Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Knockdown, Phospho-proteomics, Inhibition, Control, Negative Control